Impaired frequencies and function of platelets and tissue remodeling in chronic Chagas disease

Abstract

Chronic inflammation, as a consequence of the persistent infection with Trypanosoma cruzi, leads to continuous activation of the immune system in patients with chronic Chagas disease. We have previously shown that increased sera levels of soluble P-selectin are associated with the severity of the cardiomyopathy distinctive of chronic Chagas disease. In this study, we explored the expression of biomarkers of platelet and endothelial activation, tissue remodeling, and mediators of the coagulation cascade in patients at different clinical stages of chronic Chagas heart disease. The frequencies of activated platelets, measured by the expression of CD41a and CD62P were decreased in patients with chronic Chagas disease compared with those in uninfected subjects, with an inverse association with disease severity. Platelet activation in response to adenosine diphosphate was also decreased in T. cruzi-infected subjects. A major proportion of T. cruzi infected subjects showed increased serum levels of fibrinogen. Patients with severe cardiac dysfunction showed increased levels of endothelin-1 and normal values of procollagen I. In conclusion, chronic infection with T. cruzi induced hemostatic alterations, even in those patients who do not yet present cardiac symptoms.

Fig 1. Quantification of platelets in patients with chronic Chagas disease.

Platelet levels were measured by platelet counts, using an optical automated hematological counter (Fig 1A) and by the expression of CD41a (Fig 1B) determined by flow cytometry in whole blood samples. Each symbol represents the number of platelets per ml (Fig 1A) or the percentage of CD41a⁺ cells (Fig 1B). Median values are indicated by the horizontal lines. *P ≤ 0.05 and **p ≤ 0.01 calculated by an ANOVA test followed by a Bonferroni test for multiple comparisons. The oblique line indicates a significant tendency between medians calculated using a test for lineal trends. The full-black symbols represent patients belonging to stages G2 and G3.

Fig 2. Flow cytometric analysis of activated platelet surfaces.

Whole blood was labelled with CD41a, CD62P (P-selectin), CD63 monoclonal antibodies and analyzed by flow cytometry. (A) Platelets were identified by their characteristic side-scatter properties (granularity: x-axis) and positive labeling with a platelet-specific mAb reagent (CD41a-FITC: y-axis). (B) Histogram plots overlaid by unstained, uninfected controls and G3 patient CD62P-PE (x-axis) fluorescence of the events within the “R1” gate were further analyzed. (C) Percentage of CD41a⁺CD62P⁺ and (E) CD41a⁺CD63⁺ cells and the mean fluorescence intensity (MFI) of the (D) CD41a⁺CD62P⁺ and (F) CD41a⁺CD63⁺ expression in each of the chronic Chagas disease patients with different clinical forms of the disease studied. Median values are indicated by the horizontal lines. *P < 0.05 and was calculated by Mann-Whitney test. The oblique line indicates a significant tendency between medians calculated using a test for lineal trends. The symbols in black represent patients belonging to stages G2 and G3.

Fig 3. Determination of platelet activation in whole blood of patients with chronic Chagas disease.

The percentages of platelets before and after the addition of ADP were measured by flow cytometry. (A) The symbols represent the expression of CD41a⁺CD62P⁺ at the basal levels or after the activation with ADP. ***P <0.001 vs the nonstimulated uninfected healthy group and calculated by using a paired t test. (B) Activation index (expression of CD41a⁺CD62P⁺ cells after activation/basal expression of CD41a⁺CD62P⁺ cells) in each of the subjects evaluated. **P < 0.001 and calculated by using the Mann-Whitney U test.

Fig 4. Association between platelet activation and age in patients with chronic Chagas disease and uninfected healthy subjects.

Correlation analysis between the levels of activated platelets and age was performed by the Pearson correlation test in T. cruzi-infected subjects (A, n = 24; C, n = 14) and uninfected healthy subjects (B, n = 17; D, n = 18).

Fig 5. Plasma levels of von Willebrand factor and fibrinogen in patients with chronic Chagas disease.

The symbols represent the concentration of (A) von Willebrand factor and (B) fibrinogen for each individual evaluated in the different clinical groups determined. The dotted line shows the maximum reference value (normal range of von Willebrand = 50–160%; normal range of fibrinogen = 200–400 mg/dl). The symbols in black represent patients belonging to stages G2 and G3.

Fig 6. Levels of endothelin-1 in patients with chronic Chagas disease.

Plasma endothelin-1 levels were determined by capture ELISA. The symbols represent the concentration of endothelin-1 for each individual evaluated. The horizontal lines indicate the values of the medians for each clinical group established by the Kuschnir classification, as indicated in Materials and Methods. HF, patients with heart failure of origin not related to Chagas disease. *P ≤ 0.05 and was calculated by the unpaired t test.

Fig 7. Procollagen levels in patients with chronic Chagas disease.

The levels of procollagen peptides were measured by capture ELISA. The symbols represent the concentration of procollagen for each individual evaluated in the different clinical groups determined by the Kuschnir classification, as indicated in Materials and Methods. The horizontal lines show the median. HF, patients with heart failure unrelated to Chagas disease. *P ≤ 0.05 and was calculated by the unpaired t test. The oblique line indicates a significant tendency between medians using a test for lineal trends; P = 0.0006.